Methods for lowering blood cholesterol and compositions of alkali metal 2-methoxyestra-1, 3, 5(10)-trien-3-ol-17-one sulfate



United States Patent 3,274,056 METHODS FOR LOWERING BLOOD CHOLESTER- OL AND COMPOSITIONS 0F ALKALI METAL 2- METHOXYESTRA 1,3,5()-TRIEN-3-0L-17-0NE SULFATE Edward Warren Cantrall, New City, N.Y., Ruddy Littell, Rivervale, N.J., and Seymour Bernstein, New City, .Y., assignors to American Cyanamid Company, Stamford, Conn., a corporation of Maine No Drawing. Filed Feb. 12, 1965, Ser. No. 432,376

6 Claims. (Cl. 167-65) be illustrated by the following formula:

wherein M is an alkali metal.

The invention relates to compositions containing alkali metal salts of 2-methoxyestra-1,3,5(l0)-trien-3-ol-17-one sulfates and edible carrier. These alkali metal salts are white crystalline solids, soluble in water, partly soluble in polar organic solvents such as methanol, and generally insoluble in non-polar organic solvents such as benzene, diethyl ether, petroleum ether and the like.

The active components of this invention may be prepared, for example, by reacting Z-methoxyestrone with a sulfating agent such as chlorosulfonic acid or ethyl chlorosulfonate. The reaction is ordinarily carried out in an inert organic solvent such as methylene chloride in the presence of a base such as pyridine. The resulting pyridinium salt may be dissolved in methanol and converted into the desired salt by neutralization with sodium hydroxide or the like.

The compounds of this invention may also be prepared, for example, by reacting 2-methoxyestrone with a sulfating agent such as sulfamic acid. The reaction is ordinarily carried out in the presence of a base such as pyridine. The resulting pyridinium salt may be converted into the desired salt by neutralization with sodium hydroxide, potassium hydroxide or the like.

The steroid components described above may be dispensed as the active ingredient in compositions of the steroid and an edible carrier. While the amount of steroid to be given daily will depend on many factors such as size, weight, age, etc., of the warm-blooded animal, it has been found, for example, that a daily intake of from 1 mg. to 10 mg. of body weight will produce good results. The dosage unit may be in a form for a single unit per day, or smaller forms for use as multiple units per day. In the case of tablets, they may be of larger size, scored for use as fractional units one or more times per day.

The compositions can be dispensed in the form of soft or hard shell gelatin capsules. Also present in the capsules may be diluents such as lactose, starch, magnesium oxide, magnesium stearate and the like. The capsules may be large enough to provide the desirable daily dosage to be used in multiple doses per day.

The present compositions may be dispensed as parenteral solutions or suspensions. If larger doses in small amounts are desirable it may be necessary to use parenteral suspensions because the solubility of the steroids in substantially aqueous solutions is limited.

Compositions using the salts of the present invention as the active component may take the form of syrups or pediatric drops. Such formulations: usually contain one or more of the following suspending agents, buffer salts, stabilizers, preservatives and the like.

The steroid compounds of this invention are effective in lowering blood cholesterol in animal tests and are therefore useful in treating hypercholesteremia.

The following tested were conducted male rats, C.F.E. strain, l20140 g. initial weight, obtained from Carworth Farms, New City, New York, in a group of four was fed the compound incorporated in the diet at a 0.005% dose level for 6 days. By a heart puncture technique serum blood samples were obtained and cholesterol levels.

were determined by the Leffier procedure (Amer. J. Clin. Path, 31, 310 (1959)).

An estrogenic assay (three-day assay) was performed in the intact immature female rat (4060 g. Sherman strain) essentially according to B. L. Rubin et al, Endocrinology, 49, 429 (1951). The following table summarizes the above testing results:

The following example describe the compounds of the present invention.

Example 1 .Preparation of sodium Z-methoxyeslra- 1,3,5(10) -trz'en-3-0l-17-one sulfate (sodium Z-methoxyestrone sulfate) To an ice cold solution of 1.0 ml. of chlorosulfonic acid in 20 ml. of methylene chloride is added dropwise, with swirling, 15 ml. of pyridine. To the above solution is added a solution containing 1.35 g. of Z-methoxyestrone in a mixture of 20 ml. of pyridine and 40 ml. of methylene chloride. The resulting mixture is allowed to stand at room temperature for three'days. The mixture is filtered and the filtrate is concentrated to about 20 ml. Ether (ca. ml.) is added and the mixture is cooled. The pyridinium salt (410 mg., melting point 205209 C.) precipitates, is filtered, suspended in methanol and is neutralized with 1 N sodium hydroxide in methanol. The resulting turbid solution is filtered and diluted with about 200 ml. of ether. The product precipitates as an amorphous white powder, 400 mg. (20% melting point 175 C.

Example II Following the procedure of Example I and neutralizing the pyridinium salt with potassium hydroxide solution produces potassium 2-methoxyestra-1,3,5(l0)-trien- 3-ol-17-one sulfate.

In a similar manner the other alkali metal salts are prepared.

Example IlI.--Preparatin 0 Potassium 3-hydroxy-2- methoxyestra-1,3,5(10) -trien-17-0ne sulfate (potassium Z-methoxyestrone sulfate) In a 2 ml. of pyridine (reagent grade) there is dissolved 500 mg. of 2-methoxyestrone and the solution is warmed in an oil bath (8595 C.) with stirring. To this is added 540 mg. of pulverized sulfamic acid. The mixture is heated at 8595 C. for 2 hours with continued stirring and is then cooled at room temperature. To the cooled mixture is added ml. of anhydrous ether, the solid material is mixed well with the ether and then collected by filtration. The solid is washed twice with anhydrous ether (5 ml. each time) and dried in vacuo over phosphorous pentoxide at room temperature for /2 hour. The solid (wt. 1.16 g.) is ground in a mortar and added to a mixture of 8 ml. of 12% aqueous potassium hydroxide and 4 ml. of pyridine. After mixing thoroughly, the organic layer is separated and filtered to remove traces of solid. The solid and mixing vessel are Washed twice with 0.5 ml. of pyridine which is added to the filtrate. To the filtrate is added 25 ml. of anhydrous ether and the resulting solid is separated from the ether by decantation and washed three times with m1. portion of anhydrous ether. To the residue is added 100 ml. of 95% ethyl alcohol (95 ml. absolute ethyl alcohol, 5 ml. water) and the solution is treated with charcoal while boiling and filtered while hot through diatomaceous earth. Upon cooling a white crystalline solid gradually separates and crystallization is completed overnight at 5 C. The solid is collected, washed with cold absolute ethanol and dried at room temperature over fresh phosphorus pentoxide at 0.5 mm. for 24 hours. There is obtained 470 mg. of a white crystalline solid, melting point ca. ZOO-250 C. dec. (66% yield).

Analysis.-Calcd. for C H O SK- /2H O: C, 53.38; H, 5.66; S, 7.50; K, 9.15, K.F., 2.11. Found: C, 52.89, 53.00; H, 5.79, 5.60; S, 7.35, 7.75; K, 9.18, 9.03; K.F., 2.15, K.F., 2.01.

S (gr-avimetric) Kl (Karl Fischer H20 determination) We claim:

1. A method of lowering blood cholesterol in warmblooded animals which comprises administering to said animal from 1 mg. to 10 mgs. per kg. of body weight of an alkali metal 2-rnethoxyestra-l,3,5(10)-trien3-ol-17- one sulfate.

2. A method of lowering blood cholesterol in warmblooded animals which comprises administering to said animals from 1 mg. to 10 mgs. per kg. of body weight of potassium 2-methoxyestra-1,3,5 l0)-trien-3-ol-1'7-one sulfate.

3. A method of lowering blood cholesterol in warmblooded animals which comprises administering to said animals from 1 mg. to 10 mgs. per kg. of body weight of sodium 2-methoxyestra-1,3,5 (10)-trien-3-ol-17-one sulfate.

4. A therapeutic composition effective in lowering blood cholesterol in warm-blooded animals which cornprises from 1 mg. to 10 mgs. per kg. of body Weight of an alkali metal 2-methoxyestra-1,3,5(10)-trien-3-ol-17-one sulfate and an edible carrier.

5. A therapeutic composition effective in lowering blood cholesterol in warm-blooded animals which comprises from 1 mg. to 10 mgs. per kg. of body weight of potassium 2-methoxyestra-1,3,5 (10)-trien-3-ol-17-one sulfate and an edible carrier.

6. A therapeutic composition effective in lowering blood cholesterol in warm-blooded animals which comprises from 1 mg. to 10 mgs. per kg. of body weight of sodium 2-methoxyestra-1,3,5(10)-trien-3-ol-17-one sulfate and an edible carrier.

No references cited.

JULIAN S. LEVITT, Primary Examiner.

VERA C. CLARKE, Examiner. 

1. A METHOD OF LOWERING BLOOD CHOLESTEROL IN WARMBLOODED ANIMALS WHICH COMPRISES ADMINISTERING TO SAID ANIMAL FROM 1 MG. TO 10 MGS. PER KG. OF BODY WEIGHT OF AN ALKALI METAL 2-METHOXYESTRA-1,3,5(10)-TRINE-3-OL-17ONE SULFATE. 